The GGDEF-EAL protein CdgB from Azospirillum baldaniorum Sp245, is a dual function enzyme with potential polar localization

Azospirillum baldaniorum Sp245, a plant growth-promoting rhizobacterium, can form biofilms through a process controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP). A. baldaniorum has a variety of proteins potentially involved in controlling the turnover of c-di-GMP many of which are coupled to sensory domains that could be involved in establishing a mutualistic relationship with the host. Here, we present in silico analysis and experimental characterization of the function of CdgB (AZOBR_p410089), a predicted MHYT-PAS-GGDEF-EAL multidomain protein from A. baldaniorum Sp245. When overproduced, CdgB behaves predominantly as a c-di-GMP phosphodiesterase (PDE) in A. baldaniorum Sp245. It inhibits biofilm formation and extracellular polymeric substances production and promotes swimming motility. However, a CdgB variant with a degenerate PDE domain behaves as diguanylate cyclase (DGC). This strongly suggest that CdgB is capable of dual activity. Variants with alterations in the DGC domain and the MHYT domain negatively affects extracellular polymeric substances production and induction of swimming motility. Surprisingly, we observed that overproduction of CdgB results in increased c-di-GMP accumulation in the heterologous host Escherichia coli, suggesting under certain conditions, the WT CdgB variant can behave predominantly as a DGC. Furthermore, we also demonstrated that CdgB is anchored to the cell membrane and localizes potentially to the cell poles. This localization is dependent on the presence of the MHYT domain. In summary, our results suggest that CdgB can provide versatility to signaling modules that control motile and sessile lifestyles in response to key environmental signals in A. baldaniorum.

Introduction Sequence alignments were performed using Clustal Omega [23] and STRAP [24]. The structural model for PAS-GGDEF-EAL of CdgB for schematic representation was built by I-TAS-SER and SwissModel servers [25,26] taking as reference the structure of RbdA (Protein Data Bank [PDB]:5XGB). The structures of the GGDEF and EAL domains for molecular coupling analyses were built by the SwissModel server taking as reference the structures of RbdA (Protein Data Bank [PDB] code 5XGD) [27] for the GGDEF domain and MucR ([PDB] code 5M1T) [28] for the EAL domain, both from P. aeruginosa. The quality of the models was analyzed on the Molprobity server [29] and a subsequent structural minimization was carried out in UCSF Chimera 1.10 software [30]. The GTP and c-di-GMP substrates were taken from the PubChem database [31], to which hydrogens were added and structural minimization was performed for each in Avogadro 2.0 [32]. PDBQT files were generated in the AutodockTools tool, where Mg 2+ ion uploads were added with the SwissModel server. Molecular coupling analyses were carried out on the AutoDockTool4 platform [33]. Finally, the visualization and preparation of figures were carried out in the UCSF Chimera 1.10 program.

Construction of plasmids and variant strains
Isolation of genomic and plasmid DNA used for DNA restriction enzyme digestion, electrophoretic agarose analysis, and transformation assays was carried out according to standard protocols [38].
The ΔcdgB mutant strain was constructed by replacing the coding region of cdgB with a kanamycin resistance cassette as previously described [17]. The DNA fragments flanking cdgB (GenBank accession number WP_014199675) were amplified by PCR using the primers Fkpnaz88 and Rxhoaz88 to generate the upstream A fragment of 992 bp, and the primers Fspeaz90 and Rsacaz90 to generate the downstream B fragment of 993 bp, each of which were subsequently cloned in the pGEM-T Easy vector, through TA cloning (Promega, Madison, WI, USA), respectively and transformed in E. coli DH5α to obtain the corresponding pGEM-A and pGEM-B constructs. The A fragment was excised with KpnI and XhoI, then was ligated into pJMS-Km suicide vector previously digested with the same restriction enzymes to yield the plasmid pJMS-Km-FA which was subsequently transformed into E. coli DH5α. Both pGEM-B and pJMS-Km-FA were digested with SpeI and SacI restriction enzymes and ligated to generate the pMMS construct, which contains the ΔcdgB::km r fragment. The pMMS construct was mobilized into A. baldaniorum Sp245 by biparental mating using E. coli S17.1 as a donor strain. Transconjugants were screened on K-lactate minimal medium with kanamycin 50 μg/mL. The mutation of interest in single colonies that were resistant to Km was further confirmed by PCR and DNA sequencing analysis.
The cdgB overexpression constructs, were generated using the pMP2444 broad host-range plasmid [36]. The full-length ORF of cdgB gene, was amplified using the Forf89 and Rorf89 primers and subsequently cloned into pGEM-TEasy to obtain the construct pGEM-cdgB. The pGEM-cdgB and pMP2444 plasmids were digested with the EcoR1 restriction enzyme. The EcoRI digested fragments corresponding to cdgB and linearized pMP2444 were ligated to generate pMP-cdgB. The desired 5'-3' orientation of the insert (cdgB expressed under the lac promoter) was confirmed by restriction analysis with HindIII. Competent E. coli S17.1 cells were transformed with the pMP-cdgB plasmid. Transformed cells were used as donors in biparental matings to transfer pMP-cdgB to A. baldaniorum Sp245. The point mutation on the GGDEF motif (D456K) was introduced by inverted PCR using the Q5 Site-Directed Mutagenesis kit (New England BioLabs) following the manufacturer's instructions (using the primer pair GGDEF-F and GGDEF-R). The resulting pMP-cdgB SGDEF-SGKEF plasmid was introduced into E. coli S17.1 and subsequently transferred to A. baldaniorum Sp245 by biparental conjugation. The same strategy was used for introducing the point mutation in the EAL motif (E580A) (using the primer pair EAL-F and EAL-R). The resulting pMP-cdgB EAL-AAL plasmid was introduced into E. coli S17.1 and subsequently transferred to A. baldaniorum Sp245 through conjugation.
All plasmid constructs were sequenced to confirm the correct sequence of cdgB and its desired orientation (downstream and under the control of the lac promoter). These constructs do not have the gene that produces the LacI repressor, hence the Plac promoter is constitutively active.
To generate a cdgB::egfp translational fusion we used a PCR fusion approach designed by Yang et al. [39]. Briefly, the cdgB gene, without a stop codon, was amplified with primers cdgB-F-24 and cdgB-R-24, and the egfp reporter gene was amplified using gfp-F-24 and gfp-R-24. The primer cdgB-R-24 and gfp-F-24 have compatible overhangs that allow fusing the amplicons through PCR amplification using the primer pair cdgB-F-24-gfp-R-24. The fused PCR product and plasmid pMP2444 were digesting with BamH1 and XbaI restriction enzymes, and ligated to generate the plasmid pMP-cdgB::egfp. E. coli S17.1 competent cells were transformed with pMP-cdgB::egfp and a transformant was used to transfer the plasmid to A. baldaniorum Sp245 by conjugation to as previously described [16].
The MHYT domain was deleted by digesting the pMP-cdgB::egfp construct with SalI. The digested fragment of 7113 bp that lack the MHYT domain was ligated to produce the pMP-cdgB ΔMHYT plasmid, which was introduced to E. coli S17.1 by chemical transformation. The plasmid was transferred by conjugation to A. baldaniorum Sp245. The cdgB ΔMHYT deletion allele was sequenced to verify that the open reading frame was not shifted.

Analysis of growth curves
To determine possible effects of plasmid pMP-cdgB or ΔcdgB deletion mutant on growth rates, growth curves were performed and compared with strains carrying the empty vector pMP2444 or WT strain. Overnight cultures were diluted to an optical density at 600 nm (OD 600 ) of 0.01 in 100 ml Erlenmeyer flasks (3 replicates per strain) containing 25 ml of NFB � medium supplemented with Gm, when it was necessary for plasmid selection. Cultures were incubated in a rotary shaker (150 r.p.m.) at 30˚C and OD 600 measured every 2 hours, using an EON microplate spectrophotometer (BioTek, Winooski, VT, USA) at 595 nm.

Determination of extracellular polymeric substance production
To analyze EPS production, A. baldaniorum and derivative strains were grown in LB � and diluted to obtain bacterial suspensions with an OD 600 of 1.2-1.4 in NFB � medium supplemented with KNO 3 and grown for five days at 30˚C, under static conditions. Next, cultures were centrifuged at 10,000 g, the pellets were suspended in 1 ml NFB � media, as described above, and a 0.005% (w/v) Congo Red (CR) colorant solution (Sigma-Aldrich, Chemical) was added to achieve a 40 μg/ml concentration. The cells were incubated with agitation (200 rpm) for two hours. Afterward, CR bound to cells was quantified as previously described [17,19]. CR measurements were normalized by total protein concentration measured with the Bradford method. The results are of three independent assays with three biological determinations.

Motility assay
The swim motility assay was performed as previously described [19,42]. Briefly, bacteria were grown in LB � medium at 30˚C until reaching 5x10 6 -5x10 7 CFU/mL, afterwards 5 μL of the culture were spotted over semisolid minimal K medium supplemented with malate, succinate, or proline (10 mM) as a carbon source and containing 0.25% (w/v) agar. The size of the bacterial motility ring was measured in cm after incubation at 30˚C for 48 h.

Relative quantification of c-di-GMP accumulation using a genetic biosensor
C-di-GMP levels were analyzed using the riboswitch-based dual fluorescence reporter system as described previously [18,19,43]. This biosensor expresses AmCyan and TurboRFP, both fluorescent proteins (green and red, respectively), from the same constitutive promoter. Tur-boRFP production is inhibited at low levels of c-di-GMP due to the presence of c-di-GMPbinding riboswitch. For this purpose, the constructs pGEX-CdgB, pGEX-cdgB SGDEF-SGKEF , pGEX-cdgB EAL-AAL , pGEX-CdgA (positive control) [17] and the empty vector pGEX-4T-1 (negative control) were used to transformed a competent E. coli S17.1 strain containing the pDZ-119 vector [37]. All bacterial strains were grown in LB medium at 30˚to an OD 600 of approximately 0.6. Afterwards, 0.1 mM IPTG was added to the bacterial culture, and these were further incubated for 24 h for induction of protein expression. Cultures were concentrated 10-fold and resuspended in water. c-di-GMP production was analyzed macroscopically relating color intensity with the production of the second messenger. Microscopic assessment of c-di-GMP was performed using a Nikon Eclipse TE2000U fluorescence microscope. A drop of the induced culture was deposited on a coverslip and covered with a 1% agarose pad. The excitation and emission of the calibrator AmCyan fluorophore were recorded at 457 and 520 nm, respectively. The reporter TurboRFP fluorophore was excited at 553 nm, while its emission was measured at 574 nm. Images obtained were edited with the Nikon NIS Elements. Merge images represented the overlay of the fluorescence images AmCyan green, TurboRFP red and both yellow. The relative fluorescence intensity (RFI) was calculated as the ratio between the TurboRFP and AmCyan fluorescence intensities and is directly proportional to cdi-GMP levels, as analyzed using ImageJ software. The RFI values represent the standard deviations of three biological replicates, and significant differences are indicated at � P < 0.05 according to Student´s t-test by SigmaPlot as previously described [19,44].

Microscopy studies
To visualize bacteria by fluorescence and confocal laser scanner microscopy (CLSM), A. baldaniorum Sp245 and its derivative strains were grown in 5 mL of NFB � medium at 30˚C, with agitation (150 rpm) for 18 h. Then, 1 mL of each culture was centrifuged at 8,000 rpm for 2 min, and each pellet was suspended in 100 μL of FM4-64FX dye (10 μg/mL) (Thermo Fisher Scientific) in phosphate buffer saline (PBS) and maintained for 10 min at 4˚C to stain the lipids of the membrane. FM4-64FX fluorescence was observed with a microscope (TE 2000U; Nikon, Tokyo, Japan) with a 100x objective lens (Plan Oil immersion) and photographed with a DS-QilMc camera (Nikon). Subsequently, 10μL of each suspension was mounted onto a glass coverslip and sealed with a 1% (w/v) agarose plug. When necessary, we also used DAPI to stain nucleoids. For video recordings, the samples were viewed with an Eclipse Ti-E C2+ confocal laser scanning microscope (Nikon) with a 60X objective lens (Plan Apo VC, water immersion). eGFP was excited at 488 nm, and its fluorescent emission was captured at 510 nm, FM4-64FX was excited at 565 nm and its fluorescent emission was captured at 734 nm, while DAPI was excited at 358 nm and its fluorescent emission was captured at 461 nm. Image slices were visualized and processed using the NIS Elements software (Nikon). The images were edited with ImageJ software (NIH, Bethesda, MD, USA) as previously described [16]. To visualize biofilms formed by the strains under study, the bacteria were grown in NFB � +KNO 3 medium supplemented with 85 μM of Calcofluor-White colorant (CWC) (Sigma-Aldrich, United States) and inoculated onto FluoroDish glass-bottom dishes (Fisher Scientific), at 30˚C in static conditions as previously described [41].
Biofilms forming on the surfaces of dishes were recorded after 5 days and observed by CLMS. CWC was excited at 440 nm with a UV laser and its emission was captured at 500-520 nm. The samples were scanned at an x/y scanning resolution of 1,024 × 1,024 pixels. The step size in the z-direction was 0.1 μm. The image stacks were visualized and processed using NIS Elements and edited using ImageJ as previously described [41]. This analysis allowed generating a three-dimensional view of the biofilm through the measurement of signal intensity. The biofilm structure can be observed as an intensity surface plot, where the intensity of the signal represents the density of EPS produced.

Western blotting analysis
For analysis of the expression of GFP-fusion proteins in A. baldaniorum Sp245, derivative strains were cultured in NFB � +KNO 3 medium at 30˚C to an OD 600 of 1.0. Bacteria cells were collected by centrifugation at 10,000 rpm for 5 min. The cells were resuspended in PBS, supplemented with Roche Complete Mini EDTA-free protease inhibitor, and ultrasonicated for 2 min. Cellular debris was sedimented by centrifugation (30 min. at 10,000 X g). The supernatant (soluble fraction) was obtained, and the pellet (insoluble fraction) was resuspended in PBS supplemented with detergent mix (0.5% Triton X 100; 0.1% SDS, and 0.5% 7-Deoxycholic acid sodium salt), and centrifuged at 25,000 X g for 2 h, to obtain protein membrane total extract. The total protein concentration of the respective fractions was determined using the Bradford method. The samples were resuspended in 5X loading buffer, boiled, separated on 10% SDS-PAGE, and subsequently transferred onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany) for immunoblotting using anti-GFP antibody (GFP D5.1 Rabbit mAb, HRP Conjugate, Cell Signaling Technology, Danvers, Massachusetts, USA.) HRP-DAB substrate Kit (Thermo Scientific Pierce) was used to check the target proteins on the membrane according to the manufacturer's guidelines.

Statistical analysis
Means were compared by Student's t-test to determine statistically significant differences. Differences were considered significant at P-values less than 0.05.

The domain architecture of CdgB contains multiple sensory domains and conserved GGDEF and EAL domains
CdgB (AZOBR_p410089) from A. baldaniorum Sp245 [45] is 794 amino acid residues long and is predicted to have seven transmembrane regions, a PAS domain, a GGDEF and an EAL domain (Fig 1A and 1B). The transmembrane regions conform a conserved MHYT domain which proposed to be involved in gas sensing [46,47] (Fig 1C). Three MHYT motifs, characterized by conserved methionine, histidine, tyrosine, and threonine residues, are located in positions 64-67, 125-128, and 187-190 (Figs 1B and S1). Each MHYT motif spans two transmembrane helices, and is projected to the outer face of the cytoplasmic membrane [46]. The PAS domain faces the cytoplasm and is comprised by 107 amino acid residues from position 256 to 362. The C-terminus of CdgB resides in the cytosol and includes both the GGDEF (363-534 aa) and EAL (544-786 aa) domains (Fig 1B).
The primary sequence of the GGDEF domain of CdgB was aligned to the GGDEF domain of two characterized DGCs, PleD and RbdA [27,48]. CdgB has an SGDEF motif in place of the canonical GGD[E]EF motif. This motif has been found in active DGCs in both bacteria and eukaryotes [49][50][51]. PleD and RbdA have an allosteric autoinhibitory site with a RXXD motif, CdgB has a PXXD motif instead, hence it is most likely not autoinhibited by its catalytic product (Fig 2A and 2B).
The amino acid sequence of the EAL domain of CdgB was aligned to the sequence of the EAL domain of two well characterized PDEs, RocR and MucR [28,52]. The alignment revealed that the EAL domain of CdgB conserve the residues involved in binding to c-di-GMP (Y 565 , Q 566 , P 567 , R 584 , D 698 , D 720 ), Mg 2+ (E 580 , N 365 , E 667 , D 697 , K 718 , E 757 , Q 774 ), and for the formation and stability of loop 6 (E 670 ). This loop is an essential structure for dimerization [53] (Fig 3A and 3B).
To extend our in silico analysis of CdgB, we conducted tertiary structure predictions using available crystal structures of validated GGDEF and EAL domains from the Protein Data Bank repository. The GGDEF domain from CdgB was modeled using as template the crystallographic coordinates of the GGDEF domain from RbdA (PDB input: 5XGD; identity 31.84%) from P. aeruginosa [27]. The EAL domain from CdgB was modeled using as template the coordinates of the three-dimensional structure of the EAL domain of MucR (PDB input: 5M1T; identity 45.82%) from P. aeruginosa [28] (Figs 2 and 3).
The three-dimensional model of the GGDEF domain of CdgB was used to analyze the potential interaction with its substrates using molecular coupling analyses (molecular docking) (Fig 2C). Our analysis predicted an interaction of GTP with the GGDEF domain of CdgB with ΔG = -8.2 Kcal/mol (Fig 2D). The interaction interphase includes residues N 420 , D 429 , D 455 . These amino acids, conserved in GGDEF domains, have been shown to be essential for the activity of DGCs [27]. The first 2 residues are important for the binding of GTP, while D 455 (located at the active site) coordinates the Mg +2 ion to perform the nucleophilic attack on the  Those that interact with cofactor are marked in green. The residue that stabilizes structurally the loop 6 is shown in alpha phosphate of GTP eliminating a pyrophosphate, resulting in the formation of c-di-GMP from another GTP molecule [48,54]. Molecular docking analysis also predicted that the EAL domain of CdgB can bind c-di-GMP with a ΔG = -8.58 Kcal/mol (Fig 3C and 3D). The N 635 residue is responsible for coordinating Mg 2+ ions and is essential for the EAL domain, as its mutation was reported to significantly affect PDE activity [28,53]. The amino acid residues R 584 and Q 566 could be involved in substrate binding by interacting with the anionic phosphate oxygen and guanine moieties of c-di-GMP [52,54,55]. These analyses suggest that CdgB could potentially act as a dual DGC/PDE protein.
We found cdgB orthologues in different species within the Azospirillum genus, as well as in other alpha-proteobacteria (S2 Fig). This could suggest that this domain architecture and potential bifunctional activity may give these bacteria a selective advantage or the ability to adapt to changes in environmental signals.

Analyses of phenotypical consequences of alterations in cdgB in A. baldaniorum Sp245
The role of GGDEF-EAL proteins with dual activity has been poorly explored, hence we decided to investigate whether cdgB influences the biofilm formation, EPS production, and swimming motility of A. baldaniorum Sp245. To do so we generated an A. baldaniorum Sp245 strain with a deletion cdgB (A. baldaniorum ΔcdgB) and use different overexpression constructs to analyze the effect of overproducing the WT allele (pMP-cdgB) and mutated alleles with different point mutations that result on altered GGDEF or EAL motifs (pMP-cdgB SGDEF-SGKEF and pMP-cdgB EAL-AAL respectively), or a deletion of the MHYT domain (pMP-cdgB ΔMHYT ), we also included the A. baldaniorum pMP-cdgB::egfp to analyze the stability of the CdgB::eGFP fusion. As negative controls, we analyzed strains containing the broad host-range plasmid pMP2444 ( Table 1).
We first evaluated the growth rate of the strains of interest in NFB � media supplemented with KNO 3 as a nitrogen source [18,19]. Our results showed that all the strains exhibited similar growth characteristics (S3 Fig). Next, we analyzed the results from biofilm formation assays. The ΔcdgB mutant strain, made 25% less biofilm compared to the parental strain (Fig  4). This could suggest that in the WT background CdgB is required for biofilm formation. Since c-di-GMP is required for biofilm formation this result would suggest that a baseline levels of expression CdgB may act as a DGC. The presence of the complementation construct pMP-cdgB in the ΔcdgB mutant strain partially restored biofilm formation compared to the WT strain with the overexpression plasmid pMP2444 (Fig 4). Interestingly, the overproduction of CdgB, CdgB-ΔMHYT and CdgB-SGDEF-SGKEF in the WT background of A. baldaniorum Sp245, resulted in decreased biofilm formation compared to both the parental WT strain and the ΔcdgB mutant strain. This suggests that, in the presence of an intact endogenous copy of cdgB, overproduction of CdgB from a plasmid has a negative effect on biofilm formation. This would indicate that at levels beyond a yet not defined threshold, CdgB may act as a PDE. To further evaluate the potential PDE activity of CdgB, we overproduced a CdgB variant with point mutations in the EAL domain that alter amino acids required for its catalytic yellow. The catalytic residue is in red. The EAL motif is marked with a red box, and loop 6 is inside a box with black outline. (B) Structural model of the CdgB EAL monomer generated by homology modeling. The EAL motif is highlighted in red and loop 6 is highlighted in orange. (C) Structural model of the CdgB EAL monomer in complex with c-di-GMP. The EAL domain is presented in surface mode. (D) Model of the EAL domain in complex with c-di-GMP generated by molecular coupling with a ΔG = -8.58 Kcal/mol and overlapped with the 5M1T crystal structure, corresponding 744 to EAL domain from P. aeruginosa showed in light purple color with an RMSD of 0.757 of ligands. The Mg 2+ ions are shown in green.

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that, when overproduced, CdgB can act as a DGC when the amino acids required for PDE activity are altered. Together these results led us to propose that CdgB is a dual-function enzyme capable of synthesizing and degrading c-di-GMP.
To further support our conclusions, we next estimated the ability of these strains to produce extracellular polymeric substance (EPS) that are likely required for biofilm formation. The strains were grown in NFB � + KNO 3 and tested for their capacity to bind the colorant Congo Red (Fig 5), a dye that binds extracellular DNA, several exopolysaccharides, amyloid proteins and has been used as an indirect measurement of c-di-GMP production [56,57]. In general, we observed that the biofilm phenotype of the different strains positively correlates with EPS production (Figs 4 and 5), except for the strains that overproduce CdgB-ΔMHYT and CdgB-SGDEF-SGKEF. We did not observe significant differences in EPS production in strains overproducing these CdgB variants compared to the control strain (no CdgB overproduction). These variants may have reduced PDE activity although more evidence is required to make that conclusion.
We further analyzed exopolysaccharide production on mature biofilms formed under static conditions in NFB � media supplemented with KNO 3 by staining with the dye calcofluor white (CWC) (Fig 6). This dye shows affinity to polysaccharides with β-1,3 and β-1,4 linkages, such as those present in capsular polysaccharides and exopolysaccharides produced by Azospirillum [58]. The ΔcdgB mutant strain and the CdgB-overexpressing-strain showed a low CWC binding, compared to WT and control strains in mature biofilms. In contrast, the strain overproducing CdgB EAL-AAL produced more exopolysaccharides compared to the controls. The strain

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overproducing CdgB SGDEF-SGKEF behaved as the WT strain and the negative control, while the strain overproducing CdgB ΔMHYT behaved comparable to the strain overproducing CdgB (Fig  6). These results further support our observations on the role of CdgB in controlling biofilm formation and EPS production, as well as for the proposed importance of EPS production on biofilm maturation in A. baldaniorum Sp245 [59].
Since motility is regulated by c-di-GMP in a opposite manner compared to biofilm formation and EPS production, we next evaluated the effect of overexpressing CdgB and the mutated variants on swimming motility in the presence of a variety of chemosensory signals (malate, succinate and proline). The overproduction of CdgB promoted swimming motility in the presence of any of the tested chemoattractants, while overproduction of the CdgB variant with the AAL motif had the opposite effect. Strains overproducing the CdgB variants with either the SGKEF motif or the deletion of the MHYT domain, as well as the ΔcdgB mutant strain, showed none or modest effects on motility (Fig 7). These results further suggest that CdgB can affect phenotypes associated with the levels of c-di-GMP, and hence that it is most likely a dual-function DGC-PDE. The switch in catalytic activity could depend on its abundance in A. baldaniorum cells or the presence of signals that inhibit its PDE activity.

CdgB has DGC activity in Escherichia coli
To analyze the effect of CdgB on c-di-GMP accumulation we examined the cellular levels of cdi-GMP with the use of a c-di-GMP biosensor in the heterologous host E. coli S17.1 [18]. When the concentration of the second messenger increases, TurboRFP increases proportionally [37,43]. To this end we analyzed CdgB native and CdgB variants with point mutations in the DGC domain (CdgB SGDEF-SGKEF ) and the EAL domain (CdgB EAL-AAL ), respectively. The expression of CdgB (pGEX-CdgB/ pDZ-119 strain) resulted in accumulation of red fluorescent individual cells and the formation of red fluorescent colonies (Fig 8). These traits were also observed when the previously characterized DGC CdgA was overproduced, but not when the empty plasmid pGEX-4T1 was used instead (Fig 8) [17]. The bacterial cells containing pGEX-CdgB SGDEF-SGKEF expression vector showed the same color as that of the negative pGEX-4T1 control. Whereas the pGEX-CdgB EAL-AAL strain increased the accumulation of red fluorescent bacterial cells and colonies (Fig 8). These results strongly suggest that CdgB has both DGC and PDE activities in E. coli.

CdgB is potentialy polarly localized
Studies have shown that some proteins involved in c-di-GMP metabolism need an specific localization to regulate cell behavior [60][61][62]. Bioinformatic analyses predicted that CdgB contains an MHYT domain with seven transmembrane helices (Fig 1). These observations prompted us to evaluate if CdgB is anchored to the cell membrane through its MHYT domain. To do so, we analyzed the localization of a CdgB-eGFP fusion protein through fluorescence microscopy. To better delineate the cellular membrane, we stained the cells with a membrane lipid-specific dye (FM4-64FX). We observed CdgB anchored to the cell membrane and eventually arrive to cell poles (Fig 9) (S1 Video). The membrane anchor of CdgB depended on the presence of the MHYT domain (Fig 9).
We also confirmed through immunoblot analysis that the strain of A. baldaniorum expressing CdgB ΔMHYT produced a truncated CdgB-eGFP protein of 85 kDa in the cytoplasmic fraction, while the A. baldaniorum strain expressing CdgB-eGFP produced a protein of 112 kDa, mainly detected in protein extracts obtained by detergent solubilization (S4 Fig). Our results unveiled that CdgB is possibly polarly localized. We found, in both strains tested (A. balda-niorum+cdgB::egfp and A. baldaniorum ΔcdgB+cdgB::egfp), that CdgB-eGFP displayed unipolar, bipolar and multisite distributions (S5 Fig). This compartmentalization could play a role in controlling the activity of this bifunctional enzyme perhaps through a signal sensed by the MHYT domain in the periplasm.

Discussion
A. baldaniorum Sp245 is an environmental bacterium capable of establishing mutualistic relationships with a variety of plants. Both as a soil dwelling bacterium and a symbiont, A. baldaniorum Sp245 is exposed to a plethora of signals that need to be integrated by sensory modules to adapt its behavior and increase fitness. Signaling modules that incorporate the second messenger c-di-GMP are crucial for this bacterium to engage in sessile or motile lifestyles [2,[17][18][19]63]. Here we reported our findings regarding the characterization of a GGDEF-EAL protein with PAS and MHYT sensory domains which we named CdgB. Proteins with GGDE-F-EAL tandem domains can be grouped based on the conservation of the GGDEF and EAL motifs. Some of these proteins have only one of the two motifs conserved [47], both domains degenerated [64,65], or both motifs conserved [10,47,66]. Typically, even when both motifs are conserved, these proteins have a predominant catalytic activity. Evidence has been accumulating of the role of bifunctional GGDEF-EAL proteins with dual enzymatic activity (DGC and PDE). The predominant activity of these proteins is controlled by self-made or external signals [67][68][69]. Our results strongly suggest that CdgB is a bifunctional enzyme. In the genetic background of A. baldaniorum Sp245, under the conditions tested, overproduction of CdgB results in phenotypic changes typically associated with the activity of PDEs. Interestingly, in a heterologous host the predominant activity observed was of DGC. These differences could stem from signals that are species-specific or the result of the growth conditions used for these experiments. We speculate that the different behavior of CdgB in E. coli could be due to differences in nitrogen metabolism. Recently Park et al. [70] demonstrated that a nitrite transporter stimulated biofilm formation by controlled NO production via appropriate nitrite reductase suppression and subsequent diguanylate cyclase activation, and c-di-GMP production in E. coli and P. aeruginosa. Additionally, E. coli possesses a transcription factor named NorR with a non-heme iron center to sense NO [71], indication of different regulation mechanisms. More work needs to be done to elucidate what could be favoring the DGC activity of CdgB in E. coli.
The sensory domain MHYT could potentially play an important role in controlling the enzymatic activity of CdgB. The first characterized hybrid protein DGC-PDE with an MHYT domain was YkoW from Bacillus subtilis although the involvement of this domain in signal perception and enzymatic function could not be ascertained. Nevertheless, it was proposed that the MHYT domain could potentially sense O 2 , CO, or NO through the coordination of one or two copper atoms [46]. Other proteins characterized to date with an MHYT domain are NbdA and MucR from P. aeruginosa both implicated in NO-mediated biofilm dispersion. The deletion of nbdA and mucR impaired NO-induced biofilm dispersion, only NbdA appeared to be specific to the NO-induced dispersion response. Although MucR was shown to play a role in biofilm dispersion in response to NO and glutamate, exposure to NO in the presence of MucR but the absence of NbdA did not result in increased PDE activity. MucR is one of the few proteins harboring both EAL and GGDEF domains that possess both DGC and PDE activity [47,66]. Furthermore, MucR is proposed to interact with the alginate biosynthesis protein Alg44, which contains a c-di-GMP binding PilZ domain essential for alginate biosynthesis [5,66]. Recently, other MucR homologous protein was described in Azotobacter vinelandii implicated in the alginate biosynthesis too [72].
Oxygen and nitric oxide are key signals during the establishment of A. baldaniorum Sp245 as a symbiont [2,40]. Interestingly, there are at least two chemotaxis receptors, Tlp1 and Aer, involved in O 2 sensing that incorporate c-di-GMP detection through a conserved PilZ domain [2,4]. Aer also integrates other chemical cues produced by the roots [4]. These chemosensory receptors could be potential signaling partners of CdgB which could perhaps sense similar chemotaxis signals. The PAS domain of CdgB could add another layer to its sensory repertoire. This domain is present in a variety of bacterial signaling proteins; and is able to bind several molecules, such as oxygen and Flavin adenine dinucleotide (FAD) [9,73]. NO has been shown to promote root growth promotion [74] and biofilm formation in A. baldaniorum Sp245, the absence of a periplasmic nitrate reductase (Nap) significantly affects biofilm formation through a mechanism yet to be explored [40]. Since the MHYT domain has been proposed to sense NO it is tempting to speculate that CdgB could participate in a singling module that controls biofilm formation in response to nitric oxide. We speculate that NO may be sensed by the MHYT domain of CdgB. Reception of this signal may influence CdgB activity by modulating DGC activity and possibly PDE domain activity. Nevertheless, the molecular details of this mechanism remain largely unknown, and need more analyses.
The polar localization of CdgB is intriguing. It remains to be shown if this recruitment occurs in the flagellated cell pole and who are the interacting partners of CdgB. Cellular compartmentalization of signaling modules opens the possibility of localized sensing and short-range signal transduction to a closely localized effector. Future work will be aimed to identify potential members of the CdgB signaling module.  ::egfp, B) A. baldaniorum ΔcdgB+cdgB::egfp were grown in NFB � medium. Photographs of the GFP fusion protein in WT and ΔcdgB strains were detected using a fluorescence microscope (TE 2000U; Nikon). Different subcellular locations of protein CdgB-eGFP including polar, bipolar, multisite were visualized. In red is showed membrane lipids with FM4-64FX, in green the CdgB-eGFP fusion, and blue bacterial nucleoid staining with DAPI. The images are representative of three biology repeats. Scale bar correspond to 10 μm. (TIF) S1 Video. Visualization of A. baldaniorum cdgB::egfp. Time-lapse. The samples were scanned at an x/y scanning resolution of 1,024 x 1,024 pixels.

Supporting information
Step size in z directions was 0.05 μm. The Plan Apo VC 60X WI objective was used. The eGFP was excited at 488nm, and the FM4-64FX was excited at 561 nm with objective lens WI (water immersion). (AVI) 39. Yang